The present invention discloses a procedure for cloning the gene encoding a DNA polymerase-exonuclease of Streptococcus pneumoniae. In its broader application, the present invention discloses a procedure for cloning and characterizing genes that encode proteins with nuclease activity in the Streptococcus pneumoniae host/vector system. As used in this invention, nuclease is an enzyme capable of hydrolizing internucleotide linkages of a nucleic acid. Exonuclease is an enzyme that hydrolyzes only a terminal phosphodiester bond of a nucleic acid. DNA polymerase is an enzyme catalyzing a template-dependent synthesis of DNA from its deoxyribonucleoside 5'-triphosphate precursors.
Bacteria generally contain several different DNA polymerase enzymes. In the best characterized case--gram-negative Escherichia coli--three enzymes are found: PolI, which is present in the highest molar concentration, and has been implicated in DNA repair; PolII, for which no function is known; and PolIII, which is responsible for chromosomal DNA synthesis. All three enzymes exhibit 3' to 5' exonuclease activity, and PolI and PolIII also exhibit 5' to 3' exonuclease activity [extensively reviewed in Kornberg, A., DNA Replication, W. H. Freeman and Co., San Francisco (1980) and Minkley et al., J. Biol. Chem., 259:10386-10392 (1984)].
Three DNA polymerases were also found in gram-positive Bacillus subtilis. Like E. coli, the PolI polymerase of B. subtilis is the predominant enzyme (and implicated in DNA repair), and PolIII functions in chromosomal replication. PolIII exhibits exonuclease activity, but only in the 3' to 5' direction. PolII does not show exonuclease activity, and the exonuclease activity of PolI has not been clarified yet.
On the other hand, the predominant polymerase in gram-positive Streptococcus pneumoniae exhibits exonuclease activity after only partial purification [Lacks, S., J. Bacteriol., 101:373-383 (1970)]. Cloning of the gene for this polymerase, PolA, was accomplished by an approach that depended upon the exonuclease activity of the enzyme, a procedure far different from that used for cloning the E. coli gene [Kelley et al., PNAS, U.S.A., 74:5632-5636 (1977)]. As detailed below, the pneumococcal gene is cloned in a gram-positive cloning system using a broad host-range multicopy plasmid vector [based on a procedure disclosed in Stassi et al., PNAS, U.S.A., 78:7028-7032 (1981)]. Unique features of the approach include the screening of clones for exonuclease activity by a DNase colony assay, and the characterization of the product of the cloned gene by a method for detecting nuclease activity in polycrylamide gels after electrophoresis and in the presence of SDS. Previously these techniques have been used for the study and characterization of proteins with nuclease activity, but never for detecting DNA polymerases. As most of the known DNA polymerases show exonuclease activity, this procedure can be generally applicable to the cloning of genes that codify for DNA polymerases, and this method is more rapid and convenient than the one used for the cloning of the E. coli polA gene.